There is a rising demand for chiral compounds in various industries, including the pharmaceutical, agrochemical, food and beverage, cosmetic, diagnostic and research industries, among others. Although considerable progress in chiral chemistry has been achieved in recent years, chemists still face many challenges in the area of stereoselective synthesis. Selectivity in a chiral synthesis reaction is important in order to achieve a high yield of a chiral compound having the desired stereochemistry. Stereochemistry is important because different stereoisomers of chiral compound (e.g. enantiomers and diasteriomers) can have very different functions. For instance, one enantiomer in a racemic drug mixture may be entirely responsible for the therapeutic effects of a drug in the body. It is therefore often desirable to produce the single stereoisomer of interest for a given application. Despite the high demand for stereoselective chiral molecules, their productivity has been low. Process and cost limitations often preclude stereoselective synthesis. Therefor, in many cases, racemic mixtures are used. Manufacturers are therefore looking for more rapid, efficient, and less expensive ways to produce stereospecific chiral molecules. Biocatalysis reactions are currently being explored over conventional chemical catalysis for selective production of chiral molecules. Biocatalysis reactions exploit enzymes, which are protein-based molecules that catalyze biological reactions. Enzymes typically display three types of selectivity that make them desirable. They are chemoselective, meaning that they act on a specific type or range of functionality, such that other sensitive functionalities in the reaction mixture (that may be targeted under chemical catalysis) are spared, resulting in a cleaner reaction. They are regioselective, meaning that, due to their complex three-dimensional structure, they may distinguish between functional goups which are located in different regions of a substrate molecule. They are enantioselective, meaning that they can recognize chirality in a substrate and tend to preferentially transform prochiral molecules into molecules having a specific chirality. Biocatalysts can often enable chiral compound synthesis in fewer steps and with lower solvent usage than conventional chemical methods. Added advantages of biocatalysts are that they are environmentally acceptable, being completely degraded in the environment, and tend to act under mild conditions, which minimizes problems of undesired side-reactions that often plague traditional chemical methodology.
One enzyme of interest as a biocatalyst is alcohol dehydrogenase (ADH). ADH enzymes are a family of enzymes that catalyse reactions to produce aldehydes, ketones, and alcohols and therefore have commercial and industrial importance. Chiral alcohols can be important building blocks in a variety of high-value chemicals including, but not limited to, pharmaceuticals, agrochemicals, and various other chiral compounds. Some ADHs preferentially catalyze the oxidation of alcohols to aldehydes and ketones, while others catalyze the reverse of such reaction, for example, to produce alcohols for biofuels.
Commercially available enzymatic catalysts typically have several shortcomings that prevent their use in industrial applications. Narrow substrate specificities, poor solvent tolerance and instability at high temperature prevent many ADHs from being used in industrial-scale applications. For instance, certain classic ADH molecules, such as yeast ADH and horse liver ADH, although inexpensive and readily available, react on very few types of alcohols and are very unstable at higher temperatures (>50° C.). ADHs from other microorganisms (e.g. E. coli and Z. mobils) and hyperthermophiles (e.g. S. solfataricus, A. pernix, T. brockii and T. ethanolicus) have been considered for large-scale chiral compound biosynthesis. However, low enzymatic activities and dependence on expensive cofactors (e.g. NADP and NADPH) and have been barriers for broad adoption in commercial-scale production processes.
Alcohol dehydrogenases are ubiquitous in three life domains and represent a family of oxidoreductases that catalyze the NAD(P)H-dependent interconversion between alcohols and the corresponding aldehydes or ketones. Interconversions of alcohols, aldehydes, and ketones are essential processes in both prokaryotes and eukaryotes. Among ADHs, the medium chain ADHs have been studied extensively, which usually contain zinc. Zinc-containing ADHs constitute a large protein family with various enzyme activities, including alcohol dehydrogenase, polyol dehydrogenase and cinnamyl alcohol dehydrognease activities. A large number of zinc-containing ADHs including those from the hyperthermophiles Pyrococcus horikoshii, Aeropyrum pernix and Sulfolobus solfataricus, contain one catalytic zinc and one structural zinc (Esposito et al. 2002; Guy et al. 2003; Ishikawa et al. 2007). The zinc-containing ADHs from mesophile Clostridium beijerinckii, and thermophiles Thermoanaerobacter brockii and Thermoanaerobacter ethanolicus contain only catalytic zinc.
Hyperthermophiles are a group of microorganisms growing optimally at ≧80° C., of which anaerobic heterotrophs have attracted increasing attention for use in fermentation reactions at elevated temperatures. All members of genus Thermococcus are chemoorganotrophs which can grow on peptide-containing substrates, and some of them are able to grow on carbohydrates including starch and chitinas as carbon source. It is demonstrated that glycolysis from glucose to pyruvate in Thermococcus celer and Thermococcus litoralis, which appears to occur via a modified EM pathway containing ADP-dependent hexose kinase and phosphofructokinase, and a tungsten-containing glyceraldehyde-3-phosphate: ferredoxin oxidoreductase. Among Thermococcus species, Thermococcus strain ES1 was firstly reported to produce ethanol under S0-limiting conditions (Ma et al. 1995). Moreover, other ADHs have been purified and characterized, all of which are iron-containing (Antoine et al. 1999; Li and Stevenson 1997; Ma et al. 1994; Ma et al. 1995).
In addition to interests in their physiological roles in production of alcohols, zinc-containing ADHs from hyperthermophiles are highly desired as promising catalysts in industrial applicaitons because of the features such as solvent tolerance, stereoselectivity as well as thermostability. In hyperthermophilic archaea, the zinc-containing ADHs from aerobic archaea S. solfataricus and A. pernix have been extensively studied in terms of structure, catalysis, function or regulation. It is known that a zinc-containing ADH from anaerobic archaeon Pyrococcus furiosus underwent asymmetric ketone reduction to the corresponding chiral alcohols. The crystal structure of a zinc-containing ADH from P. horikoshii has been resolved recently. However, no zinc-containing ADHs from Thermococcus species have been previously reported.